Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

Abstract

In the tricarboxylic acid (TCA) cycle, NADP+-specific isocitrate dehydrogenase (NADP+-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP+ as a cofactor. We constructed an NADP+-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP+-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP+-ICDH activity. Therefore, NADP+-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

Phenotypes of gene disruptants in relation to a putative mitochondrial malate–citrate shuttle protein in citric acid-producing Aspergillus niger

“…Strains of A. niger and Escherichia coli, cultivation conditions and media. A. niger WU-2223L, 6,11,13,[37][38][39][40][41][42] a citric acid-producing strain was used as a parental strain which has been deposited in National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) (accession number NBRC 111403; http://www.nite.go.jp/). As described later, strain DCTPA-1 (a ctpA disruptant strain) and cCTPA-1 (a strain DCTPA-1 complemented with ctpA) were used.…”

“…Measurement of intracellular organic acids from the mycelia of A. niger strains and calculation of the intracellular volume were performed according to the methods described previously. 11) Determination of mycelial dry weight, citric acid, and glucose in the culture broth.…”

“…Mycelial dry weights, citric acid, and glucose in the culture broth were determined basically according to the methods described previously. 11) The culture filtrate was recovered by filtration of the culture broth through glass microfiber filter GF/A and qualitative paper filter no. 4.…”

“…[3][4][5][6] Because citric acid is an intermediate of the tricarboxylic acid (TCA) cycle, the causal relationships between citric acid production and the activities of related enzymes in TCA cycle and glycolysis have been extensively studied. [6][7][8][9][10][11][12][13] On the other hand, the transport system exporting citric acid generated in mitochondria must be also a very important subject of research directly linked to citric acid production. Guebel and Torres predicted that the transport of citric acid from mitochondria is one of the major regulatory steps of citric acid production in A. niger using a metabolic flux balance model.…”

Biobased organic acids production by metabolically engineered microorganisms

“…Citric acid is a successful example of a commercially bioproduced organic acid with annual production estimated at 1.75 million tons in 2011 and having a continuous increasing demand/consumption [5]. Besides its predominant use in the food and beverage industry, citric acid has been also used in pharmaceuticals and a range of technical and industrial segments as well.…”